A myriad of biomarkers containing aldehyde and ketone moieties exist and can play an important role in the biological, forensic, medical and industrial sciences. In particular, aldehydes and ketones are known to be key end products in the degradation of a variety of biological molecules, such as, lipids, nucleic acids, carbohydrates and proteins. In a number of instances, these end products are a result of oxidative stress. In one example, malondialdehyde and 4-hydroxynonenal are markers for lipid peroxidation.
A number of reagents for the detection of aldehyde and ketone-containing moieties have been proposed, however, each with limited success. Among the most widely used of such reagents are dansyl hydrazine, fluorescein thiosemicarbazide, various biotin hydrazides, biotin hydroxylamine (ARP), and various aromatic amines (2-aminopyridine, 8-aminonaphthalene-1,3,6-disulfonic acid, 1-aminopyrene-3,6,8-trisulfonic acid, 2-aminoacridone). Unfortunately, these reagents require additional purification and/or secondary reagents.
Existing methods of labeling carbohydrates that utilize hydrazine, hydroxylamine and amine derivatization reagents have focused on labeling aldehydes present in, or introduced into, carbohydrates, particularly the so-called “reducing sugars”. Aldehydes are typically introduced into carbohydrates by periodate oxidation. The adduct formed with the reducing sugar can be further stabilized by treatment with borohydride or a cyanoborohydride. The derivatization reaction typically proceeds or is followed by a separation technique such as chromatography, electrophoresis, precipitation, affinity isolation or other means before direct or indirect detection of the labeled product. Unlike the foregoing, which require purification and the use of a secondary detection reagent, the reagents of the present invention permit rapid in-situ detection of aldehyde and ketone moieties upon contact.
U.S. Pat. No. 6,967,251 (Haugland et al.) describes aniline-substituted quinazolinone compounds which can display fluorescent changes upon binding of aldehyde-containing compounds in a gel. However, the aniline-substituted quinazolinone core, which is important for binding of the analyte, is less fluorescent and fluorogenic than the compounds of the present invention. Accordingly, the present invention provides substantial advantages over the previously described aldehyde and ketone detector compounds.
A family of hydrazinyl substituted xanthene dyes have been previously described in U.S. Pat. No. 4,420,627 (Widiger). However, each of the xanthene moieties in Widiger are substituted to prevent analyte binding of the molecule. Particularly, the terminal nitrogen atom in the hydrazinyl moiety in Widiger is carbonylated, such that the hydrazinyl group is not nucleophilic and unable to bind an aldehyde or ketone moiety. Accordingly, the compounds are not functional in the analyte detection method of the present invention, which involve a quenched molecule up until the analyte binding event. Accordingly, the compounds in Widiger are functionally and structurally very different from those of the present invention.